Use of Soluble Forms of CD83 and Nucleic Acids Encoding them for the Treatment or Prevention of Diseases

ABSTRACT

The present invention provides for the use of soluble forms of CD83 and nucleic acids encoding them for the treatment of diseases caused by the dysfunction or undesired function of a cellular immune response involving dendritic cells, T cells and/or B cells. The invention moreover provides soluble CD83 molecules specifically suited for said purpose, antibodies against said specific soluble CD83 proteins and assay methods and kits comprising said antibodies.

This is a continuation of U.S. patent application Ser. No. 10/535,522,filed Apr. 13, 2006, now allowed, which is a 371 of PCT/EP03/12941 filedNov. 19, 2003, which claims foreign priority benefit under 35 U.S.C.§119 of European Patent Application No. 02025851.3 filed Nov. 19, 2002.

The present invention provides for the use of soluble forms of CD83 andnucleic acids encoding them for the treatment of diseases caused by thedysfunction or undesired function of a cellular immune responseinvolving dendritic cells, T cells and/or B cells. The inventionmoreover provides soluble CD83 molecules specifically suited for saidpurpose, antibodies against said specific soluble CD83 proteins andassay methods and kits comprising said antibodies.

BACKGROUND OF THE INVENTION

The immune system of mammals must possess the capability to react to avery large number of foreign antigens. Lymphocytes constitute a centralelement of the immune system because they can recognize antigens andeffect a specific, adaptive immune response. Lymphocytes can be dividedinto two general classes of cells, B-lymphocytes, which are capable ofexpressing antibodies, and T lymphocytes that can be sub-divided intoCD4+ helper T cells and CD8+ cytotoxic T cells. Both of these sub-groupsof T lymphocytes are capable of recognizing antigens associated withsurface proteins known as the major histocompatibility complex (MHC).The recognition of the MHC occurs throughout the T cell receptor (TCR),a protein complex that is anchored in the cytoplasmic membrane of Tcells. The CD8+ T cell receptor exclusively mediates interactionsbetween MHC class I antigens and cytotoxic T cells; the CD4+ T cellreceptor exclusively mediates interactions between MHC class II antigensand helper T cells.

The triggering of an immune response does not exclusively progress fromT cells alone, but rather, through the interaction of T cells withso-called antigen presenting cells (APCs, also known as accessory cells)and their surface markers (for example MHC II).

These accessory cells can be sub-divided into “simple” APCs whosefunction is to present antigens and “professional” APCs that, besidefrom presenting antigens, also have an accessory function in stimulatinglymphocytes. APCs themselves do not have antigen specificity but serveas “nature's adjuvant” by presenting antigens to T cells. Aside frommononuclear phagocytes, dendritic cells (DC) are members of the APCtype. In fact, DCs are the most potent APC known today and they are theonly APC that are also able to stimulate naive T cells and are thereforecalled “natures adjuvants”. As a result of their differentcharacteristics and function, two types of dendritic cells have beenclassified to date:

follicular dendritic cells (also known as lymphoid-related DCs) that arepresent in the lymph nodes, spleen and mucosa-associated lymph tissuesand interdigitating dendritic cells (also known as myeloid derived DCs)that are found in the interstitial space of most organs, in T cell richzones of the lymph nodes sand spleen and are distributed throughout theskin where they are known as Langerhans cells.

Immature dendritic cells, i.e. DCs that are not fully capable ofstimulating T cells, have the function of taking up antigens andprocessing them into MHC-peptide complexes. Stimuli such as TNF-alpha(tumor necrosis factor) and CD40L induce the maturation of dendriticcells and lead to a massive de novo synthesis of MHC class I and MHCclass II molecules and to a migration of the DC, for example, from theinterstitial space of the internal organs through the blood into thelymph nodes of the spleen and liver. Moreover, increased expression ofco-stimulator molecules (for example, CD80, CD86) and adhesion molecules(for example, LFA3) occurs during the migration phase into the secondarylymphoid tissues. Mature DC stimulate T lymphocytes upon arrival in theT cell rich regions of the secondary lymphoid tissue by presentingpeptide antigens within the context of MHC class I or MHC class II tothese T cells. Depending on the conditions, DCs can stimulate theactivation of a variety of T cells which, in turn, can bring about adifferential response of the immune system. For example, as mentionedabove, DCs that express MHC class I can cause cytotoxic T cells toproliferate and DCs that express MHC class II can interact with helper Tcells. In the presence of mature DCs and the IL-12 that they produce,these T cells differentiate into Th1 cells that produceinterferon-gamma.

Interferon-gamma and IL-12 serve together to promote T-killer cells. Inthe presence of IL-4, DCs induce T cells to differentiate into Th2 cellswhich secrete IL-5 and IL-4 that in turn activates eosinophils andassist B cells to produce antibodies (Banchereau, J. and Steinman, R. M.(1998) Nature 392:245-252).

DCs can also induce a so-called mixed leukocyte reaction (MLR) in vitro,a model for allogenic T cell activation and graft rejection.

A typical feature of these MLR-assays is the formation of large DC-Tcell-clusters. Addition of hCD83ext at day 1 strongly inhibited thetypical cell cluster formation of DC and proliferating T cells(Lechmann, M. et al. (2001) J. Exp. Med. 194:1813-1821).

Mature DC characteristically express, amongst others (e.g. MHC I and II,CD80/86, CD40) the marker molecule CD83 on their cell surface (Zhou,L.-J. and Tedder, T. F. (1995) J. Immunology, vol. 154:3821-3835). Thisis one of the best markers for mature DC known today.

CD83, a molecule from the Ig superfamily of proteins, is a single chain,43 kDa glycoprotein consisting of 205 amino acids (SEQ ID NO:2) in itsimmature form. The first 19 amino acids represent the signal peptide ofCD83 and they are lost upon insertion of the protein into the membrane,leaving a 186 amino acid membrane spanning protein. The mature CD83 hasan extracellular domain formed by amino acids 20 to 144 (SEQ ID NO:2), atransmembrane domain comprising amino acids 145 to 166 (SEQ ID NO:2),and cytoplasmic domain formed by amino acids 167 to 205 (SEQ ID NO:2).The extracellular domain has as structural feature a single Ig-like(V-type) domain, and is expressed very strongly on the cell surface ofmature DC. The extracellular domain of the CD83 protein differs from thetypical Ig-like domain in that it is encoded by at least two exons: oneexon only codes for a half of the Ig-like domain, whereas the other exonencodes the membrane spanning domain (see Zhou, L.-J., Schwarting, R.,Smith, H. M. and Tedder, T. F. (1999) J. Immunology, vol. 149:735-742).The cDNA encoding human CD83 contains a 618 bp open reading frame (SEQID NO:1, see Genbank ID: Z11697 and Zhou, L.-J. et al, supra (1995)).

While the precise function of CD83 remains to be determined, it has beendemonstrated that inhibition of CD83 cell surface expression on matureDC by interference with nuclear export of CD83 mRNA leads to a clearreduction in the capacity of these cells to stimulate T cells. (Kruse,M. et al. (2000) J. Exp. Med. 191:1581-1589). Thus, CD83 appears to berequired for DC function.

Furthermore it was found that when a soluble form of CD83 wasadministered to cells, the amount of CD83 expressed by the cells wasreduced (mature dendritic cells) or the cells did not start to produceCD83 (immature dendritic cells). Since immature dendritic cells have noCD83 in/on their membrane, this observation lead to the conclusion, thatsoluble CD83 must interact with another cell (membrane) protein thanCD83, i.e. a heterophilic interaction is suspected to occur between thesoluble CD83 and an unidentified ligand (Lechmann, M. et al. (Dec. 17,2001) J. Exp. Med. 194:1813-1821 and (June 2002) Trends in Immunology,Vol. 23(6):273-275). Evidence for the occurrence of soluble CD83 in vivoalso exist. Soluble CD83 has been found in normal human sera and seemsto be released from activated dendritic cells and B-lymphocytes (Hock etal. (2001) Int. Immunol. 13:959-967).

WO 97/29781 relates to methods and compositions (vaccines) forstimulating a humoral immune response in which a soluble form of CD83 isemployed as an adjuvant together with a given antigen. Soluble formscomprise CD83 fusion protein and a soluble form consisting of aminoacids 1 to 124, the extracellular domain of CD83. In addition to the useof CD83 as adjuvant for vaccine preparations, this document discussesthe use of antagonists (antibodies) against CD83 for inhibitingundesirable antigen specific responses in mammals.

WO 93/21318 describes a CD83 protein here designated HB15, chimeric HB15molecules and HB15 fragments including a fragment consisting of theextracellular domain (amino acids 1 to 125) of HB15. Furthermoreantibodies against HB15 are mentioned. However, neither a potential usenor a function of said antibodies is given. Because of the role of HB15as an accessory molecule for lymphocyte activation, the soluble HB15 andfragments is proposed to be useful as an agonist for augmentation of theimmune response. Again, no experimental proof is provided.

U.S. Pat. No. 5,710,262 and the corresponding WO 95/29236 reveal humanand mouse HB15 as potentially useful drug in the treatment of AIDS (withregard to the DNA and amino acid sequence of mous HB15, see SEQ ID Nos:3and 4). The extracellular domain of HB15 as described therein comprisesthe first 19 amino acids of the signal peptide, followed by 106 aminoacids of the extracellular domain.

The above-mentioned WO 93/21318 and WO 95/29236 also emphasize thatmonoclonal antibodies against CD83 are suitable for removing endogenousCD83 or monitor CD83 levels in serum.

It was surprisingly found that the extracellular domain of CD83(hereinafter also “hCD83ext”) comprising amino acids 20 to 144 (SEQ IDNO:2), can engage in heterophilic interactions with ligands on dendriticcells. Since the current literature only describes completeextracellular domains or extracellular domains lacking amino acids fromthe C-terminus of the extracellular domain (U.S. Pat. No. 5,710,262, WO95/29236 and WO 97/29781) it was also surprising that hCD83ext adoptedthe correct confirmation, allowing interactions with dendritic cells. Ofeven greater surprise was the effect hCD83ext had on dendritic cells; itprevented maturation of immature dendritic cells and reduced theexpression of CD83 in mature dendritic cells. As a result dendriticcells lost their ability to activate T cells. Thus, the soluble hCD83extitself was shown to be suitable for the treatment or prevention ofdiseases or medical conditions caused by undesirable immune responses,in particular by preventing activation of T cells. hCD83ext was alsofound suitable for the treatment or prevention of diseases or medicalconditions caused by undesirable immune responses mediated by dendriticcells, T cells and/or B cells.

Recently it was found that due to the fact that the hCD83ext possessesthe correct conformation of natural CD83, it is also suitable orpreparing antibodies against CD83 (see Lechmann et al., ProteinExpression and Purification 24, 445-452 (Mar. 5, 2002)). Said articlealso discloses the cloning of the extracellular domain of CD83 and theisolation of a CD83 fragment comprising amino acids 23 to 128.

Moreover, it was found that the amount of soluble CD83 protein in thehuman serum varies and is significantly higher in case of tumors andB-cell leukemia. Thus, antibodies against the soluble CD83 protein arepowerful tools for determining certain diseases (such as tumor,autoimmune diseases, viral infection, etc.) in a patient.

Finally it was found that hCD83ext exists in a monomeric and homodimerform (both being comparatively active) and that the replacement of oneor more of the cysteine residues, in particular of the fifth cysteine bya different amino acid residue (e.g. by a serine residue) in theextracellular domain of hCD83ext leads to a monomeric extracellular CD83molecule which is not susceptible to spontaneous dimerization.

SUMMARY OF THE INVENTION

Extraordinarily, soluble hCD83ext can engage with immature and maturedendritic cells, preventing maturation of the immature dendritic cells.Furthermore, mature dendritic cells treated with soluble hCD83ext arecompletely inhibited in their T cell stimulatory activity. Thus T cellsdo not proliferate anymore. CD83 has been recognized as a marker formature dendritic cells capable of T-cell (and also B cell) interaction.Formerly mature and active dendritic cells treated with soluble hCD83extare unable to form clusters with T cell (and B cells) in vitro. Hencethe dendritic cells cannot induce anymore the division/stimulation of Tcells.

As a result, the invention provides the use of a soluble form of amember of the CD83 family of proteins are suitable for the treatment orprevention of a disease or medical condition caused by the dysfunctionor undesired function of a cellular immune response involving dendriticcells, T cells and/or B cells. In particular, the soluble forms of amember of the CD83 family of proteins inhibit the interaction betweendendritic cells and T cells and between dendritic cells and B cells.

Moreover, specific soluble CD83 proteins (including homodimers, monomersand particular substitution muteins) are provided which are suitable forthe treatment or prevention of diseases defined above. Said soluble CD83proteins were found to be particular suited for raising antibodiesagainst CD83 proteins.

Finally, the invention provides that such antibodies are suitable inassays for determining diseases correlated with an enhanced precursor ofsoluble CD83 protein in the patient's serum.

More specifically the present invention provides

(1) the use of a soluble form of a member of the CD83 family of proteins(hereinafter shortly “soluble CD83 protein”), a fragment, a dimeric formand/or a functional derivative thereof, for the production of amedicament for the treatment or prevention of a disease or medicalcondition caused by the dysfunction or undesired function of a cellularimmune response involving dendritic cells, T cells and/or B cells;(2) the use of (1) above, wherein the soluble CD83 protein is a dimer,preferably a homodimer connected through one or more of the cysteineresidues within the soluble monomeric CD83 protein;(3) the use of (1) above, wherein the soluble CD83 protein is amonomeric CD83 protein, preferably a monomeric CD83 protein where one ormore of the cysteine residues have been substituted by same or differentsmall and/or polar amino acid residues;(4) the use of (1), (2) or (3) above, wherein the medicament is suitablefor the treatment or prevention of paralysis, preferably for thetreatment or prevention of paralysis associated with progressivemultiple sclerosis;(5) the use of a nucleic acid or vector having a DNA fragment encoding aCD83 protein as defined in (1), (2) or (3) above for the production of amedicament for the treatment or prevention of a disease or medicalcondition caused by the dysfunction or undesired function of a cellularimmune response involving dendritic cells, T cells and/or B cells;(6) the use of (1) to (3) and (5) above, wherein said disease or medicalcondition caused by the dysfunction or undesired function of a cellularimmune response involving dendritic cells, T cells and/or B cells isselected from the group consisting of allergies, asthma, rejection of atissue or organ transplant, autoimmune syndromes such as myastheniagravis, multiple sclerosis, vasculitis, cronic inflammatory bowldiseases such as Morbus Crohn or colitis ulcerosa, HLA B27-associatedautoimmunopathis such as Morbus Bechterew, and systemic lupuserythematosis, skin diseases such as psoriasis, rheumatoid arthritis,insulin-dependent diabetes mellitus and AIDS;(7) a soluble form of a member of the CD83 family of proteins comprisingamino acids 20 to 144 of SEQ ID NO:2, a fragment, dimeric form and/or afunctional derivative thereof;(8) a nucleic acid or recombinant expression vector encoding the CD83protein of (7) above;(9) a dimeric soluble CD83 protein as defined in (1) or (2) above;(10) a monomeric soluble CD83 protein as defined in (3) above;(11) a nucleic acid or recombinant expression vector encoding the CD83protein of (9) or (10) above;(12) a prokaryotic or eukaryotic host cells transformed/transfected witha nucleic acid or a vector of (8) or (11) above;(13) a method for producing the soluble CD83 protein of (7), (9) or (10)above, which comprises culturing a transferred/transfected prokaryoticor eukaryotic host cell according to (12) above;(14) a pharmaceutical composition comprising the soluble CD83 protein of(7), (9) or (10) above or a nucleic acid or vector as defined in (5),(8) or (11) above;(15) an antibody against a soluble CD83 protein as defined in (7), (9)or (10) above;(16) an assay method for in vitro determining the amount of soluble CD83protein in the serum of a patient which comprises contacting a serumsample with the antibody of (15) above;(17) a kit for performing the assay method of (15) above and comprisingthe antibody of (14) above; and(18) a method for treating or preventing a disease or medical conditioncaused by the dysfunction or undesired function of a cellular immuneresponse involving dendritic cells, T cells and/or B cells comprisingadministering the person in need for such treatment a pharmaceuticallysuitable amount of the soluble CD83 protein of (7), (9) or (10) above orof a nucleic acid or vector as defined in (5), (8) or (11) above.

DESCRIPTION OF THE FIGURES

FIG. 1: Partial sequence of pGEX2ThCD83ext vector. The sequence of theextracellular CD83 domain is shown in bold letters. The amino-acidsequence “GSPG” (SEQ ID NO:14) was added to the N-terminus of theextracellular CD83 domain and is part of the thrombin cleavage sitewhich is underlined. The C-terminal amino acid “I” is part of thecytoplasmic domain of CD83. Smal and EcoRI cloning sites are indicatedby a broken line (--).

FIG. 2: Purification of hCD83ext. A-D show the chromatographic elutionprofiles of the 4 purification steps. The collected aliquots aredepicted in black. Proteins of the collected fractions wereelectrophoresed using a 15% polyacrylamid gel under reducing anddenaturing conditions and visualized with Coomassie brilliant bluestaining. In addition, D also shows Western blot analysis. A: Affinitychromatography using a GSTrap column: Lane 1: molecular weight marker(MWM); Lanes 4-10: aliquots of GST-hCD83ext. B: Anion exchangechromatography using a Source 15QPE 4.6/100 column: Lane 1: MWM; Lanes2-7 aliquots of GST-hCD83ext. C: purification of the thrombin cleavageproducts using GSTrap-affinity chromatography: Lane 1: MWM; Lanes 2-4:collected flowthrough containing the cleaved hCD83ext. D: Gel filtrationusing a Superdex 75 (26/16) column: Lane 1: MWM; Lane 2: hCD83ext. Theright panel shows the Western blot analysis using an anti-CD83 antibody.E: Lyophilization, equal amounts of CD83ext aliquots, taken before andafter freeze drying, were loaded onto a 15% SDS-PAGE.

FIG. 3: hCD83 inhibits DC maturation. FACS analysis of DC. A: immatureDC where matured in the presence of the maturation cocktail from day 5-8(=mock control for mature DC). B: immature DC where matured in thepresence of the maturation cocktail (day 5-8) and on day 7 hCD83ext wasadded for 24 hours. C: immature DC where incubated in the presence ofthe maturation cocktail in combination with hCD83 from day 5-8. On day 8cells where washed and stained with the indicated antibodies andanalyzed by FACS

FIG. 4: hCD83ext inhibits allogeneic T cell proliferation. MLR analysis:hCD83ext reduced T cell proliferation in a dose dependent manner. GST,which was purified in the same way as hCD83ext and BSA (each 5 μg/ml)were used as controls.

FIG. 5: hCD83ext inhibits murine allogeneic T cell proliferation. A: MLRanalysis: hCD83ext reduced T cell proliferation in a dose dependentmanner (concentration see FIG. 4). GST, which was purified in the sameway as hCD83ext was used as control (5 μg/ml). B: The biologicalactivity in an MLR analysis as in FIG. 5A is preserved afterlyophilization.

FIG. 6: hCD83ext inhibits murine experimental autoimmuneenzephalo-myelitis (EAE) A: in an in vivo model for multiple sclerosis(MS); B: the inhibition has a long lasting effect; and C: is suitablefor therapeutic applications (hCD83ext was given every second day(fourteen times in total), starting from day 3 after the EAE induction.

FIG. 7: SDS-PAGE of hCD83ext with and without 2-mercaptoethanol (ME).

FIG. 8: Partial sequence of pGEX2ThCD83ext_mut129_CtoS vector. Thesequence of the extracellular CD83 domain is shown in bold letters. Theexchanged nucleotide and amino acid residues are enlarged. Theamino-acid sequence “GSPG” (SEQ ID NO:14) was added to the N-terminus ofthe extracellular CD83 domain and is part of the thrombin cleavage sitewhich is underlined. The C-terminal amino acid “I” is part of thecytoplasmic domain of CD83. Smal and EcoRI cloning sites are indicatedby a broken line (--).

FIG. 9: SDS-PAGE of hCD83ext and hCD83ext_mut129_CtoS with and without2-mercaptoethanol (ME).

FIG. 10: CD83 inhibits restimulation of spleen cells after the first EAEinduction (A) and also after the second EAE induction (B).

FIG. 11: Soluble CD83 inhibits cytokine production by spleen cells afterfirst EAE induction (A) and after a second EAE induction (B)

DETAILED DESCRIPTION OF THE INVENTION

Using a PCR strategy the extracellular domain of CD83 plus the firstcodon of the cytoplasmic domain were amplified from a full-length humancDNA clone and inserted behind the gluthathione-transferase gene into anexpression vector. In the resulting fusion protein the N-terminalglutathione-transferase (GST) was separated by a thrombin cleavage sitefrom the extracellular CD83 domain extended by the Ile from thecytoplasmic domain. The fusion protein was purified from an overnightbacterial culture, subjected to thrombin cleavage and the hCD83extfurther purified. The purified hCD83ext was used in dendritic cellmaturation and T-cell stimulation (MLR) assays. Surprisingly, additionof hCD83ext to immature dendritic cells induced an altered surfacemarker expression pattern. CD80 expression was reduced from 96 to 66%and CD83 expression from 96 to 30%. Also mature dendritic cells changedthe surface marker expression pattern upon exposure to hCD83ext. CD83expression was reduced from 96 to 66%. Dendritic cells treated withhCD83ext lost their ability to stimulate T-cell proliferation. Theseresults suggested a potential use of hCD83ext for treatment of dendriticcell, T-cell and/or B cell mediated diseases and conditions. Thereforethe effects of hCD83ext on Experimental Autoimmune Enzephalomyelitis(EAE), a model for Multiple Sclerosis, were studied. Surprisingly, themice treated with hCD83ext did not develop the typical paralysisassociated with EAE.

Hence according to embodiment (1) of the invention the soluble form of amember of the CD83 family of proteins, a fragment thereof, or afunctional derivative thereof may be used for the production of amedicament for the treatment or prevention of a disease or medicalcondition caused by the dysfunction or undesired function of a cellularimmune response involving dendritic cells, T cells and/or B cells.Preferably soluble CD83 protein comprises at least amino acid residues20 to 144, or 20 to 145 of SEQ ID NO: 2. Suitable fragments are thosehaving the same activity and conformation as natural CD83. Suitablederivatives include, but are not limited to, those proteins havingadditional sequences attached to its C- or N-terminus, e.g. thosecarrying part of a transmembrane domain at their C-terminus or carryingat there N-terminus a short functional peptide (Gly-Ser-Pro-Gly (SEQ IDNO:14)) may be used. The medicaments containing these proteins andfragments are useful for the treatment or prevention of paralysis, asfor example seen with progressive multiple sclerosis.

In a similar manner, nucleic acids or vectors coding for these proteinsor fragments thereof may be used in the production of medications forthe treatment and prevention of medical conditions caused by thedysfunction or undesired function of cellular immune responses involvingdendritic cells, T cells and/or B cells. In particular DNA sequencescomprising nucleotides 58 to 432, more preferably 58 to 435 of SEQ IDNO: 1 may be used. These medicaments may be used for the downregulationon RNA and/or protein level of the expression of CD83 in mammals.

The use of these medicaments for the prevention or treatment of diseasessuch as allergies, asthma, rejection of a tissue or organ transplant,autoimmune syndromes such as myasthenia gravis, multiple sclerosis,vasculitis, cronic inflammatory bowl diseases such as Morbus Crohn orcolitis ulcerosa, HLA B27-associated autoimmunopathis such as MorbusBechterew, and systemic lupus erythematosis, skin diseases such aspsoriasis, rheumatoid arthritis, insulin-dependent diabetes mellitus andAIDS may be appropriate.

Methods of treatment and/or prevention of medical conditions caused bydysfunction or undesired T cell function may comprise administering aneffective amount of CD83 or fragments as described herein; a methodmight also comprise administering an effective amount of a nucleic acidor vector as described above; the methods might be applied for thetreatment or prevention of diseases such as allergies, asthma, rejectionof a tissue or organ transplant, autoimmune syndromes such as myastheniagravis, multiple sclerosis, vasculitis, cronic inflammatory bowldiseases such as Morbus Crohn or colitis ulcerosa, HLA B27-associatedautoimmunopathis such as Morbus Bechterew, and systemic lupuserythematosis, skin diseases such as psoriasis, rheumatoid arthritis,insulin-dependent diabetes mellitus and AIDS.

As defined herein, the term “inhibit the interaction” is used toindicate that the soluble forms of the members of the CD83 family ofproteins of the present invention are capable of disrupting theinteraction of dendritic cells to T cells and/or B cells and/orinhibiting the formation of dendritic cell-T cell clusters or dendriticcell-B cell clusters in vitro at physiological pH and saltconcentrations, preferably, at pH concentrations ranging from pH 6.0 to8.0 and/or at salt concentrations ranging from 50 mM to 250 mM,preferably 125 mM to 175 mM.

A preferred assay for determining the binding of dendritic cells to Tcells and the formation of dendritic cell-T cell clusters is provided inthe Examples (Lechmann, M. et al. (2001) J. Exp. Med. 194:1813-1821).The soluble forms of the members of the CD83 family of proteins for usein the present invention are capable of causing a disruption in thebinding of dendritic cells to T cells and/or B cells and/or theformation of dendritic cell-T cell clusters or dendritic cell-B cellclusters of at least 25%, more preferably at least 50%, still morepreferably at least 75% and most preferably at least 90% or greater asmeasured in the one of the above assays. The term “soluble form” of theCD83 family of proteins is used here to define a proteinaceous moleculethat has at least a portion of the extracellular domain of a member ofthe CD83 family of proteins, but does not have an amino acid sequencethat is capable of anchoring said molecule to the membrane of a cell inwhich it is expressed. The nucleic acid sequence encoding human CD83protein as well as the amino acid sequence of CD83 are described inZhou, L. J. et al. (1992) J. Immunol. 149(2):735-742 (Genbank accessionnumber Z11697) and are provided in SEQ ID NO:1 and SEQ ID NO:2,respectively.

As defined herein, a member of the CD83 family of proteins includes anynaturally occurring protein that has at least 70%, preferably 80%, andmore preferably 90% or more amino acid identity to the human CD83 asdepicted in SEQ ID NO:2.

Thus, aside from human CD83 itself, members of the CD83 family ofproteins include the mouse HB15 protein that is encoded by the nucleicacid sequence of SEQ ID NO:3 and is represented by the amino acidsequence provided in SEQ ID NO:4, (Genbank accession number NM_(—)009856(Berchthold et al).

Other naturally occurring members of the CD83 family of proteins can beobtained by hybridizing a nucleic acid comprising, for example, all orthe extracellular portion of the human CD83 coding region or mouse HB15coding region to various sources of nucleic acids (genomic DNA, cDNA,RNA) from other animals, preferably mammals, or from other tissues ofthe same organism.

Hybridization refers to the binding between complementary nucleic acidsequences (e.g., sense/antisense, siRNA, etc.). As is known to thoseskilled in the art, the T_(m) (melting temperature) refers to thetemperature at which the binding between sequences is no longer stable.As used herein, the term “selective hybridization” refers tohybridization under moderately stringent or highly stringent conditions,which can distinguish CD83 related nucleotide sequences from unrelatedsequences.

In nucleic acid hybridization reactions, the conditions used in order toachieve a particular level of stringency will vary, depending on thenature of the nucleic acids being hybridized. For example, the length,degree of sequence complementarity, sequence composition (e.g., the GCv. AT content), and type (e.g., RNA v. DNA) of the hybridizing regionscan be considered in selecting particular hybridization conditions. Anadditional consideration is whether one of the nucleic acids isimmobilized, for example, on a filter.

In general, the stability of a nucleic acid hybrid decreases as thesodium ion decreases and the temperature of the hybridization reactionincreases. An example of moderate stringency hybridization reaction isas follows: 2×SSC/0.1 SDS at about 37° C. or 42° C. (hybridizationconditions); 0.5×SSC/0.1% SDS at about room temperature (low stringencywash conditions); 0.5×SSC/0.1% SDS at about 42° C. (moderate stringencywash conditions). An example of high stringency hybridization conditionsis as follows: 2×SSC/0.1% SDS at about room temperature (hybridizationconditions); 0.5×SSC/0.1% SDS at about room temperature (low stringencywash conditions); 0.5×SSC/0.1% SDS at about 42° C. (moderate stringencywash conditions); and 0.1×SSC/0.1% SDS at about 65° C. (high stringencyconditions).

Typically, the wash conditions are adjusted so as to attain the desireddegree of stringency. Thus, hybridization stringency can be determined,for example, by washing at a particular condition, e.g., at lowstringency conditions or high stringency conditions, or by using each ofthe conditions, e.g., for 10-15 minutes each, in the order listed above,repeating any or all of the steps listed. Optimal conditions forselective hybridization will vary depending on the particularhybridization reaction involved, and can be determined empirically.

Once a nucleic acid encoding a naturally occurring CD83 protein has beencloned, the extracellular domain can be determined by comparison of theextracellular domain of known CD83 molecules with that of the clonedCD83 sequence. A soluble form of a given naturally occurring CD83protein can then be expressed recombinantly using the techniques asdescribed herein. For example, a nucleic acid encoding a soluble form ofCD83 can be produced, inserted into a vector and transformed intoprokaryotic or eukaryotic host cells using well known techniquesdescribed herein and further known in the art (Sambrook et al. MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.,1989).

Thus, when cloning in bacterial systems, constitutive promoters such asT7 and the like, as well as inducible promoters such as pi, ofbacteriophage X, plac, ptrp, ptac (ptrp-lac hybrid promoter) may beused. When cloning in mammalian cell systems, constitutive promoterssuch as SV40, RSV, CMV including CMV-IE, and the like or induciblepromoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., the mousemammary tumor virus long terminal repeat; the adenovirus late promoter)may be used. Promoters produced by recombinant DNA or synthetictechniques may also be used to provide for transcription of the nucleicacid sequences of the invention.

Mammalian expression systems which utilize recombinant viruses or viralelements to direct expression may be engineered. For example, when usingadenovirus expression vectors, nucleic acid of interest may be ligatedto an adenovirus transcription/translation control complex, e.g., thelate promoter and tripartite leader sequence. Alternatively, thevaccinia virus 7.5K promoter may be used.

Of particular interest are vectors based on bovine papilloma virus (BPV)which have the ability to replicate, as extrachromosomal elements.Shortly after entry of an extrachromosomal vector into mouse cells, thevector replicates to about 100 to 200 copies per cell. Becausetranscription of the inserted cDNA does not require integration of theplasmid into the host's chromosome, a high level of expression occurs.These vectors can be used for stable expression by including aselectable marker in the plasmid, such as the neo gene, for example.Alternatively, the retroviral genome can be modified for use as a vectorcapable of introducing and directing the expression of the nucleic acidof interest in host cells. High level expression may also be achievedusing inducible promoters, including, but not limited to, themetallothionein RA promoter and heat shock promoters.

In yeast, a number of vectors containing constitutive or induciblepromoters may be used. A constitutive yeast promoter such as ADH or LEU2or an inducible promoter such as GAL may be used. Alternatively, vectorsthat facilitate integration of foreign nucleic acid sequences into ayeast chromosome, via homologous recombination for example, are known inthe art and can be used.

A nucleic acid of interest encoding a soluble form of a member of theCD83 family of proteins for use according to the present invention maybe inserted into an expression vector for expression in vitro (e.g.,using in vitro transcription/translation assays or commerciallyavailable kits), or may be inserted into an expression vector thatcontains a promoter sequence which facilitates transcription and/ortranslation in either prokaryotes or eukaryotes (e.g., an insect cell)by transfer of an appropriate nucleic acid into a suitable cell. A cellinto which a vector can be propagated and its nucleic acid transcribed,or encoded polypeptide expressed, is referred to herein as a “hostcell”.

The term also includes any progeny of the subject host cell. Moreover, anucleic acid of interest according to the present invention may beinserted into an expression vector for expression in vivo for somaticgene therapy. With these vectors, for example, retroviral vectors,Adenovirus vectors, Adeno-associated virus vectors, plasmid expressionvectors, the nucleic acids of the invention are expressed uponinfection/introduction of the vector into DC.

Host cells include but are not limited to microorganisms such asbacteria, yeast, insect and mammalian organisms. For example, bacteriatransformed with recombinant bacteriophage nucleic acid, plasmid nucleicacid or cosmid nucleic acid expression vectors containing a nucleic acidof interest; yeast transformed with recombinant yeast expression vectorscontaining a nucleic acid of interest; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing a nucleic acid ofinterest; insect cell systems infected with recombinant virus expressionvectors (e.g., baculovirus) containing a nucleic acid of interest; oranimal cell systems infected with recombinant virus expression vectors(e.g., retroviruses, adenovirus, vaccinia virus) containing a nucleicacid of interest, or transformed animal cell systems engineered forstable expression.

For long-term expression of the soluble forms of members of the CD83family of proteins in host cells, stable expression is preferred. Thus,using expression vectors which contain viral origins of replication, forexample, cells can be transformed with a nucleic acid of interestcontrolled by appropriate control elements (e.g., promoter/enhancersequences, transcription terminators, polyadenylation sites, etc.).Optionally, the expression vector also can contain a nucleic acidencoding a selectable or identifiable marker conferring resistance to aselective pressure thereby allowing cells having the vector to beidentified, grown and expanded. Alternatively, the selectable marker canbe on a second vector that is cotransfected into a host cell with afirst vector containing an invention polynucleotide.

A number of selection systems may be used, including, but not limited tothe herpes simplex virus thymidine kinase gene, hypoxanthine-guaninephosphoribosyltrans-ferase gene, and the adeninephosphoribosyltransferase genes can be employed in tk-, hgprt or aprtcells respectively. Additionally, antimetabolite resistance can be usedas the basis of selection for dhfr, which confers resistance tomethotrexate; the gpt gene, which confers resistance to mycophenolicacid; the neomycin gene, which confers resistance to the aminoglycosideG-418; and the hygromycin gene, which confers resistance to hygromycin.Additional selectable genes have been described, namely trpB, whichallows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine; and ODC(ornithine decarboxylase) which confers resistance to the ornithinedecarboxylase inhibitor, 2-(difluoromethyl)-DL-onithine, DFMO.

As used herein, the term “transformation” means a genetic change in acell following incorporation of DNA exogenous to the cell. Thus, a“transformed cell” is a cell into which (or a progeny of which) a DNAmolecule has been introduced by means of recombinant DNA techniques.

Transformation of a host cell with DNA may be carried out byconventional techniques known to those skilled in the art. For example,when the host cell is a eukaryote, methods of DNA transformationinclude, for example, calcium phosphate co-precipitates, conventionalmechanical procedures such as microinjection, electroporation, insertionof a plasmid encased in liposomes, and viral vectors. Eukaryotic cellsalso can be cotransformed with DNA sequences encoding a nucleic acid ofinterest, and a second foreign DNA molecule encoding a selectablephenotype, such as the those described herein. Another method is to usea eukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein.

Following transformation, the soluble form of CD83 may be isolated andpurified in accordance with conventional methods. For example, lysateprepared from an expression host (e.g., bacteria) can be purified usingHPLC, size-exclusion chromatography, gel electrophoresis, affinitychromatography, or other purification technique. Substantially pureproteins can also be obtained by chemical synthesis using a peptidesynthesizer (e.g. Applied Biosystems, Inc., Foster City, Calif.; Model430A or the like).

According to embodiment (2) of the invention the compounds for use inthe medicament of the present invention may be a dimeric structures ofthe soluble form of CD83. Preferably the dimeric structure is ahomodimer. Dimerisation may be achieved through formation of one or moredisulfide bonds between the cysteine residues present within themonomeric form of the soluble CD83 protein (which are present at aa 12,27, 35, 100, 107, 129, 163 in SEQ ID NO:2), or by means of abifunctional linker molecule (e.g. a diamine, a dicarboxylic acidcompound or the like) connecting same or different functional moieties(e.g. carboxy groups, amino groups, hydroxy groups, thio groups, etc.)within the monomeric form of the soluble CD83 protein. The latter alsoincludes the use of polypeptide linkers (e.g. out of small polar aminoacid residues such as—[(Gly)_(x)Ser]_(y)—(where x is e.g. 3 or 4 and yis e.g. 1 to 5)) to yield dimeric structures which can directly beproduced by recombinant techniques.

Particularly preferred is a homodimer (such as a homodimer comprisingamino acid residues 20 to 144 of SEQ ID NO:2 or 1 to 130 of SEQ ID NO:8)connected via a disulfide bond between the fifth cysteine residue of thesoluble CD83 (i.e., the cysteine residue corresponding to aa 129 in SEQID NO:2 and aa 114 in SEQ ID NO:8).

The compounds for use in the present invention also include derivativesof soluble forms of members of the CD83 family of proteins according tothe invention as mentioned above in which one or more amino acids hasbeen added, deleted, substituted, inserted or inverted as long as thesederivatives remain soluble as defined above and are capable of causing adisruption in the binding of dendritic cells to T cells and/or B cellsand/or the formation of dendritic cell-T cell clusters as defined above.It also includes splice variants of the CD83 compounds mentionedhereinbefore.

Particular preferred additions are those where the soluble CD83 proteinas defined hereinbefore has one or more amino acid residues derived fromthe neighbouring intracellular domain at its C-terminus, preferably thesoluble CD83 protein comprises amino acid residues 20 to 145 of SEQ IDNO:2; and/or has functional sequences attached to its N-terminus,preferably functional sequences of up to 10 amino acid residues, andmost preferably carries at the N-terminus the additional amino acidsGly-Ser-Pro-Gly (SEQ ID NO:14).

When one or more amino acids of a soluble form of a member of the CD83family of proteins is substituted, it is preferred that the one or moreamino acids are conservatively substituted. For example, conservativesubstitutions include substitutions in which aliphatic amino acidresidues such as Met, Ile, Val, Leu or Ala are substituted for oneother. Likewise, polar amino acid residues can be substituted for eachother such as Lys and Arg, Glu and Asp or Gln and Asn.

Particular substitution muteins of the soluble CD83 protein of theinvention are those of embodiments (3) and (10) of the invention,wherein the soluble CD83 protein is a monomer CD83 protein where one ormore of the cysteine residues have been substituted by same or differentshort and/or polar amino acid residue(s). Preferably the small and/orpolar amino acid residues are selected from serine, alanine, glycine,valine, threonine, etc., preferably is serine. Moreover, it is preferredthat one cysteine residue, more preferably the fifth cysteine residue,has been replaced. Most preferably the soluble CD83 protein comprisesamino acid residues 20 to 144 of SEQ ID NO:2, where the cysteine residueat position 129 has been replaced by a serine residue, or amino acidresidues 1 to 130 of SEQ ID NO:10. Such defined monomeric moleculespossess particular importance for pharmaceutical application.

According to the invention, derivatives of a soluble form of a member ofthe CD83 family of proteins also include derivatives in which one ormore of the amino acids therein has an altered side chain. Suchderivatized polypeptides include, for example, those comprising aminoacids in which free amino groups form amine hydrochlorides, p-toluenesulfonyl groups, carobenzoxy groups; the free carboxy groups form salts,methyl and ethyl esters; free hydroxl groups that form 0-acyl or 0-alkylderivatives as well as naturally occurring amino acid derivatives, forexample, 4-hydroxyproline, for proline, 5-hydroxylysine for lysine,homoserine for serine, ornithine for lysine etc. Also included are aminoacid derivatives that can alter covalent bonding, for example, thedisulfide linkage that forms between two cysteine residues that producesa cyclized polypeptide.

A soluble form of a member of the CD83 family of proteins or derivativesthereof can have a native glycosylation pattern of a CD83 molecule or analtered glycosylation pattern or can be non-glycosylated as long asthese molecules are soluble as defined above and are capable of causinga disruption in the binding of dendritic cells to dendritic cells, Tcells and/or B cells and/or the formation of dendritic cell-T cellclusters as defined above.

In a preferred embodiment, the soluble form of CD83 for use in thepresent invention comprises amino acids 20 to amino acids 144, morepreferably amino acids 20 to 145, of the human CD83 protein as depictedin SEQ ID NO:2 or amino acids 1 to 130 of SEQ ID NO:8.

In a further preferred embodiment the soluble form of CD83 for use inthe present invention comprises amino acids 22 to amino acids 135 of themouse HB15 protein as depicted in SEQ ID NO:4.

The present invention also relates to the use of a nucleic acid or anexpression vector encoding a soluble form of a member of the CD83 familyof proteins or a derivative of such a protein for the production of amedicament for the treatment or prevention of a disease or medicalcondition caused by the dysfunction or undesired function of a cellularimmune response involving dendritic cells, T cells and/or B cells.

The nucleic acids for use in the present invention as described abovecan be in the form of DNA (deoxyribonucleic acid) which contains thebases adenine, thymine, guanine and cytosine or RNA (ribonucleic acid)which contains the bases adenine, uracil, guanine and cytosine ormixtures of the two.

When the nucleic acid molecule for use in the invention is derived fromhuman CD83 protein, the portion of the coding region is preferably fromnucleotide 58 to 432 of the sequence in SEQ ID NO:1. Alternatively, theportion of the coding region is from nucleotide 58 to 435 of thesequence in SEQ ID NO:1.

When the nucleic acid molecule for use in the invention is derived fromthe mouse HB15 protein, the portion of the coding region is preferablyfrom about nucleotide 76 to 418 of the sequence in SEQ ID NO:3.

A nucleic acid that encodes a protein for use according to the inventionmay be inserted into a vector. The term “vector” refers to a plasmid,virus or other vehicle known in the art that can be manipulated byinsertion or incorporation of a polynucleotide. Such vectors can be usedfor genetic manipulation (i.e., “cloning vectors”) or can be used totranscribe or translate the inserted polynucleotide (“expressionvectors”). A vector generally contains at least an origin of replicationfor propagation in a cell and a promoter. Control elements, includingexpression control elements as set forth herein, present within anexpression vector are included to facilitate proper transcription andtranslation (e.g., splicing signal for introns, maintenance of thecorrect reading frame of the gene to permit in-frame translation of mRNAand, stop codons etc.). The term “control element” is intended toinclude, at a minimum, one or more components whose presence caninfluence expression, and can also include additional components, forexample, leader sequences and fusion partner sequences.

As used herein, the term “expression control element” refers to one ormore nucleic acid sequence that regulates the expression of a nucleicacid sequence to which it is operatively linked. An expression controlelement operatively linked to a nucleic acid sequence controlstranscription and, as appropriate, translation of the nucleic acidsequence. Thus an expression control element can include, asappropriate, promoters, enhancers, transcription terminators, a startcodon (e.g., ATG) in front of a protein-encoding gene. “Operativelylinked” refers to a juxtaposition wherein the components so describedare in a relationship permitting them to function in their intendedmanner.

By “promoter” is meant a minimal sequence sufficient to directtranscription. Both constitutive and inducible promoters are included inthe invention (see e.g. Bitter et al., Methods in Enzymology153:516-544, 1987). Inducible promoters are activated by externalsignals or agents. Also included in the invention are those promoterelements which are sufficient to render promoter-dependent geneexpression controllable for specific cell-types, tissues orphysiological conditions; such elements may be located in the 5′, 3′ orintronic regions of the gene. Promoters useful in the invention alsoinclude conditional promoters. A “conditional promoter” is a promoterwhich is active only under certain conditions. For example, the promotermay be inactive or repressed when a particular agent, such as a chemicalcompound, is present. When the agent is no longer present, transcriptionis activated or derepressed.

A nucleic acid of interest according to the present invention may beinserted into an expression vector for expression in vivo for somaticgene therapy. With these vectors, for example, retroviral vectors,Adenovirus vectors, Adeno-associated virus vectors, plasmid expressionvectors, the nucleic acids of the invention are expressed uponinfection/introduction of the vector into dendritic cells, T cellsand/or B cells.

Furthermore, the invention relates to a method of treatment orprevention of a disease or medical condition caused by the dysfunctionor undesired function of a cellular immune response involving dendriticcells, T cells and/or B cells, wherein an effective amount of a solubleform of hCD83ext is administered to a subject.

Moreover, the invention relates to a method of treatment or preventionof a disease or medical condition caused by the dysfunction or undesiredfunction of a cellular immune response involving dendritic cells, Tcells and/or B cells, wherein an effective amount of a nucleic acid orexpression vector encoding a soluble hCD83ext is administered to asubject.

According to the invention, a soluble hCD83ext or a nucleic acid orexpression vector encoding hCD83ext can be used to treat or preventrejection of tissue and/or organ transplants, particularly xenogenictissue and/or organ transplants, that occurs as a result of for examplegraft-vs.-host disease or host-vs.-graft disease.

In a further embodiment of the present invention, a soluble form of amember of the CD83 family of proteins or a nucleic acid or expressionvector encoding a hCD83ext can be used to treat or prevent undesirableresponse to foreign antigens and therewith allergies and asthma orsimilar conditions.

Other disorders, diseases and syndromes that can be treated or preventedby the use of a soluble hCD83ext or a nucleic acid or expression vectorencoding a soluble hCD83ext include autoimmune syndromes such asmyasthenia gravis, multiple sclerosis, vasculitis, cronic inflammatorybowl diseases such as Morbus Crohn or colitis ulcerosa, HLAB27-associated autoimmunopathis such as Morbus Bechterew, and systemiclupus erythematosis, skin diseases such as psoriasis, rheumatoidarthritis, insulin-dependent diabetes mellitus and AIDS.

In particular hCD83ext is suitable for the treatment of paralysisassociated with multiple sclerosis.

For therapeutic or prophylactic use, the compounds of the presentinvention alone, or in combination with other immune modulatorycompounds, e.g. tolerance inducing antigens, are administered to asubject, preferably a mammal, more preferably a human patient, fortreatment or prevention in a manner appropriate for the medicalindication. Transcutan, intracutan, subcutan and/or systemicadministration may be chosen for the delivery of hCD83ext andderivatives thereof.

The production of pharmaceutical compositions with an amount of one ormore compounds according to the invention and/or their use in theapplication according to the invention occurs in the customary manner bymeans of common pharmaceutical technology methods. For this, thecompounds according to the invention are processed together withsuitable, pharmaceutically acceptable adjuvents and/or carriers tomedicinal forms suitable for the various indications and types ofapplication. Thereby, the medicaments can be produced in such a mannerthat the respective desired release rate is obtained, for example aquick flooding and/or a sustained or depot effect.

Preparations for parenteral use, to which injections and infusionsbelong, are among the most important systemically employed medicamentsfor the above mentioned indications.

Preferably, injections are prepared either in the form of vials or alsoas so-called ready-to-use injection preparations, for example asready-to-use syringes or single use syringes in addition to perforationbottles for multiple withdrawals. Administration of the injectionpreparations can occur in the form of subcutaneous (s.c.), intramuscular(i.m.), intravenous (i.v.), internodal (i.n.) or intracutaneous (i.e.)application. The respective suitable injection forms can especially beproduced as solutions, crystal suspensions, nanoparticular orcolloid-disperse systems, such as for example, hydrosols.

The injectable formulations can also be produced as concentrates whichcan be adjusted with aqueous isotonic dilution agents to the desireddosage of the compounds of the invention. Furthermore, they can also beproduced as powders, such as for example lyophilisates, which are thenpreferably dissolved or dispersed immediately before application withsuitable diluents. The infusions can also be formulated in the form ofisotonic solutions, fat emulsions, liposome formulations, microemulsionsand liquids based on mixed micells, for example, based on phospholipids.As with injection preparations, infusion formulations can also beprepared in the form of concentrates to dilute. The injectableformulations can also be applied in the form of continuous infusions asin stationary as well as in out-patient therapy, for example in the formof mini-pumps.

Albumin, plasma expanders, surface active compounds, organic olvents, pHinfluencing compounds, complex forming compounds or polymeric compoundscan be added to the parenteral medicinal forms with the aim ofdecreasing the adsorption of the compounds of the present invention tomaterials such as injection instruments or packaging materials, forexample plastic or glass.

The compounds according to the invention can be bound to nanoparticlesin the preparations for parenteral use, for example on finely dispersedparticles based on poly(meth)acrylates, polyacetates, polyglycolates,polyamino acids or polyether urethanes. The parenteral formulations canalso be constructively modified as depot preparations, for example onthe multiple unit principle, where the compounds of the presentinvention are incorporated in a most finely distributed and/ordispersed, suspended form or as crystal suspensions, or on the singleunit principle, where the compounds according to the invention areenclosed in a medicinal form, for example, a tablet or a seed which issubsequently implanted. Often, these implantation or depot medicamentsin single unit and multiple unit medicinal forms consist of so-calledbiodegradable polymers, such as for example, polyether urethanes oflactic and glycolic acid, polyether urethanes, polyamino acids,poly(meth)acrylates or polysaccharides.

Sterilized water, pH value influencing substances, such as for exampleorganic and inorganic acids or bases as well as their salts, buffersubstances for setting the pH value, agents for isotonicity, such as forexample sodium chloride, monosodium carbonate, glucose and fructose,tensides and/or surface active substances and emulsifiers, such as forexample, partial fatty acid esters of polyoxyethylene sorbitan (Tween®)or for example fatty acid esters of polyoxethylene (Cremophor®), fattyoils such as for example peanut oil, soybean oil and castor oil,synthetic fatty acid esters, such as for example ethyl oleate, isopropylmyristate and neutral oil (Miglyol®) as well as polymer adjuvents suchas for example gelatin, dextran, polyvinylpyrrolidone, organic solventadditives which increase solubility, such as for example propyleneglycol, ethanol, N,N-dimethylacetamide, propylene glycol or complexforming compounds such as for example citrates and urea, preservatives,such as for example hydroxypropyl benzoate and hydroxymethyl benzoate,benzyl alcohol, anti-oxidants, such as for example sodium sulfite andstabilizers, such as for example EDTA, are suitable as adjuvents andcarriers in the production of preparations for parenteral use.

In suspensions, addition of thickening agents to prevent the settling ofthe compounds of the present invention from tensides and peptizers, tosecure the ability of the sediment to be shaken, or complex formers,such as EDTA, ensues. This can also be achieved with the variouspolymeric agent complexes, for example with polyethylene glycols,polystyrol, carboxymethylcellulose, Pluronics® or polyethylene glycolsorbitan fatty acid esters. The compounds according to the invention canalso be incorporated in liquid formulations in the form of inclusioncompounds, for example with cyclodextrins. As further adjuvents,dispersion agents are also suitable. For production of lyophilisates,builders are also used, such as for example mannite, dextran,saccharose, human albumin, lactose, PVP or gelatin varieties.

A further systemic application form of importance is peroraladministration as tablets, hard or soft gelatin capsules, coatedtablets, powders, pellets, microcapsules, oblong compressives, granules,chewable tablets, lozenges, gums or sachets. These solid peroraladministration forms can also be prepared as sustained action and/ordepot systems. Among these are medicaments with an amount of one or moremicronized compounds of the present invention, diffusions and erosionforms based on matrices, for example by using fats, wax-like and/orpolymeric compounds, or so-called reservoir systems. As a retardingagent and/or agent for controlled release, film or matrix formingsubstances, such as for example ethylcellulose,hydroxypropylmethylcellulose, poly(meth)acrylate derivatives (forexample Eudragit®), hydroxypropylmethylcellulose phthalate are suitablein organic solutions as well as in the form of aqueous dispersions. Inthis connection, so-called bio-adhesive preparations are also to benamed in which the increased retention time in the body is achieved byintensive contact with the mucus membranes of the body. An example of abio-adhesive polymer is the group of Carbomers®.

For sublingual application, compressives, such as for examplenon-disintegrating tablets in oblong form of a suitable size with a slowrelease of the compounds of the present invention, are especiallysuitable. For purposes of a targeted release of compounds of the presentinvention in the various sections of the gastrointestinal tract,mixtures of pellets which release at the various places are employable,for example mixtures of gastric fluid soluble and small intestinesoluble and/or gastric fluid resistant and large intestine solublepellets. The same goal of releasing at various sections of thegastrointestinal tract can also be conceived by suitably producedlaminated tablets with a core, whereby the coating of the agent isquickly released in gastric fluid and the core of the agent is slowlyreleased in the small intestine milieu. The goal of controlled releaseat various sections of the gastrointestinal tract can also be attainedby multilayer tablets. The pellet mixtures with differentially releasedagent can be filled into hard gelatin capsules.

Anti-stick and lubricant and separating agents, dispersion agents suchas flame dispersed silicone dioxide, disintegrants, such as variousstarch types, PVC, cellulose esters as granulating or retarding agents,such as for example wax-like and/or polymeric compounds on the basis ofEudragit®, cellulose or Cremophor® are used as a further adjuvants forthe production of compressives, such as for example tablets or hard andsoft gelatin capsules as well as coated tablets and granulates.

Anti-oxidants, sweetening agents, such as for example saccharose, xyliteor mannite, masking flavors, aromatics, preservatives, colorants, buffersubstances, direct tableting agents, such as for examplemicrocrystalline cellulose, starch and starch hydrolysates (for exampleCelutab®), lactose, polyethylene glycols, polyvinylpyrrolidone anddicalcium phosphate, lubricants, fillers, such as lactose or starch,binding agents in the form of lactose, starch varieties, such as forexample wheat or corn and/or rice starch, cellulose derivatives, forexample methylcellulose, hydroxypropylcellulose or silica, talcumpowder, stearates, such as for example magnesium stearate, aluminumstearate, calcium stearate, talc, siliconized talc, stearic acid, acetylalcohol and hydrated fats are used.

In this connection, oral therapeutic systems constructed especially onosmotic principles, such as for example GIT (gastrointestinaltherapeutic system) or OROS (oral osmotic system), are also to bementioned.

Effervescent tablets or tabs, both of which represent immediatelydrinkable instant medicinal forms which are quickly dissolved orsuspended in water are among the perorally administratable compressives.Among the perorally administratable forms are also solutions, forexample drops, juices and suspensions, which can be produced accordingto the above given method, and can still contain preservatives forincreasing stability and optionally aromatics for reasons of easierintake, and colorants for better differentiation as well as antioxidantsand/or vitamins and sweeteners such as sugar or artificial sweeteningagents. This is also true for inspisated juices which are formulatedwith water before ingestion. Ion exchange resins in combination with oneor more compounds of the present invention are also to be mentioned forthe production of liquid ingestable forms.

A special release form consists in the preparation of so called floatingmedicinal forms, for example based on tablets or pellets which developgas after contact with body fluids and therefore float on the surface ofthe gastric fluid. Furthermore, so-called electronically controlledrelease systems can also be formulated by which release of the compoundsof the present invention can be selectively adjusted to individualneeds.

A further group of systemic administration and also optionally topicallyeffective medicinal forms are represented by rectally applicablemedicaments. Among these are suppositories and enema formulations. Theenema formulations can be prepared based on tablets with aqueoussolvents for producing this administration form. Rectal capsules canalso be made available based on gelatin or other carriers.

Hardened fat, such as for example Witepsol®, Massa Estarinum®, Novata®,0coconut fat, glycerol-gelatin masses, glycerol-soap-gels andpolyethylene glycols are suitable as suppository bases.

For long-term application with a systematic release of the compounds ofthe present invention up to several weeks, pressed implants are suitablewhich are preferably formulated on the basis of so-called biodegradablepolymers.

As a further important group of systemically active medicaments,transdermal systems are also to be emphasized which distinguishthemselves, as with the above-mentioned rectal forms, by circumventingthe liver circulation system and/or liver metabolism. These plasters canbe especially prepared as transdermal systems which are capable ofreleasing the compounds of the present invention in a controlled mannerover longer or shorter time periods based on different layers and/ormixtures of suitable adjuvents and carriers. Aside from suitableadjuvents and carriers such as solvents and polymeric components, forexample based on Eudragit®, membrane infiltration increasing substancesand/or permeation promoters, such as for example oleic acid, Azone®,adipinic acid derivatives, ethanol, urea, propylglycol are suitable inthe production of transdermal systems of this type for the purpose ofimproved and/or accelerated penetration.

As topically, locally or regionally administration medicaments, thefollowing are suitable as special formulations: vaginally or genitallyapplicable emulsions, creams, foam tablets, depot implants, ovular ortransurethral administration installation solutions. For opthalmologicalapplication, highly sterile eye ointments, solutions and/or drops orcreams and emulsions are suitable.

In the same manner, corresponding otological drops, ointments or creamscan be designated for application to the ear. For both of theabove-mentioned applications, the administration of semi-solidformulations, such as for example gels based on Carbopols® or otherpolymer compounds such as for example polyvinylpyrolidone and cellulosederivatives is also possible.

For customary application to the skin or also to the mucus membrane,normal emulsions, gels, ointments, creams or mixed phase and/oramphiphilic emulsion systems (oil/water-water/oil mixed phase) as wellas liposomes and transfersomes can be named. Sodium algenate as a gelbuilder for production of a suitable foundation or celluolosederivatives, such as for example guar or xanthene gum, inorganic gelbuilders, such as for example aluminum hydroxides or bentonites(so-called thixotropic gel builder), polyacrylic acid derivatives, suchas for example Carbopol®, polyvinylpyrolidone, microcrystallinecellulose or carboxymethylcellulose are suitable as adjuvents and/orcarriers. Furthermore, amphiphilic low and high molecular weightcompounds as well as phospholipids are suitable. The gels can be presenteither as hydrogels based on water or as hydrophobic organogels, forexample based on mixtures of low and high molecular paraffinhydrocarbons and vaseline.

Anionic, cationic or neutral tensides can be employed as emulsifiers,for example alkalized soaps, methyl soaps, amine soaps, sulfanatedcompounds, cationic soaps, high fatty alcohols, partial fatty acidesters of sorbitan and polyoxyethylene sorbitan, for example lanettetypes, wool wax, lanolin, or other synthetic products for the productionof oil/water and/or water/oil emulsions.

Hydrophilic organogels can be formulated, for example, on the basis ofhigh molecular polyethylene glycols. These gel-like forms are washable.Vaseline, natural or synthetic waxes, fatty acids, fatty alcohols, fattyacid esters, for example as mono-, di-, or triglycerides, paraffin oilor vegetable oils, hardened castor oil or coconut oil, pig fat,synthetic fats, for example based on acrylic, caprinic, lauric andstearic acid, such as for example Softisan® or triglyceride mixturessuch as Miglyol® are employed as lipids in the form of fat and/or oiland/or wax-like components for the production of ointments, creams oremulsions.

Osmotically effective acids and bases, such as for example hydrochloricacid, citric acid, sodium hydroxide solution, potassium hydroxidesolution, monosodium carbonate, further buffer systems, such as forexample citrate, phosphate, Tris-buffer or triethanolamine are used foradjusting the pH value. Preservatives, for example such as methyl- orpropyl benzoate (parabenes) or sorbic acid can be added for increasingstability.

Pastes, powders or solutions are to be mentioned as further topicallyapplicable forms. Pastes often contain lipophilic and hydrophilicauxiliary agents with very high amounts of fatty matter as aconsistency-giving base. Powders or topically applicable powders cancontain for example starch varieties such as wheat or rice starch, flamedispersed silicon dioxide or silica, which also serve as diluents, forincreasing flowability as well as lubricity as well as for preventingagglomerates.

Nose drops or nose sprays serve as nasal application forms. In thisconnection, nebulizers or nose creams or ointments can come to use.

Furthermore, nose spray or dry powder formulations as well as controlleddosage aerosols are also suitable for systemic administration of thecompounds of the present invention.

These pressure and/or controlled dosage aerosols and dry powderformulations can be inhaled and/or insufflated. Administration forms ofthis type also certainly have importance for direct, regionalapplication in the lung or bronchi and larynx. Thereby, the dry powdercompositions can be formulated for example as invention compound-softpellets, as an invention compound-pellet mixture with suitable carriers,such as for example lactose and/or glucose. For inhalation orinsufflation, common applicators are suitable which are suitable for thetreatment of the nose, mouth and/or pharynx. The compounds of thepresent invention can also be applied by means of an ultrasonicnebulizing device. As a propellant gas for aerosol spray formulationsand/or controlled dosage aerosols, tetrafluoroethane or HFC 134a and/orheptafluoropropane or HFC 227 are suitable, wherein non-fluorinatedhydrocarbons or other propellants which are gaseous at normal pressureand room temperature, such as for example propane, butane or dimethylether can be preferred. Instead of controlled dosage aerosols,propellant-free, manual pump systems can also be used.

The propellant gas aerosols can also suitably contain surface-activeadjuvents, such as for example isopropyl myristate, polyoxyethylenesorbitan fatty acid ester, sorbitan trioleate, lecithins or soyalecithin.

In addition, when the pharmaceutical composition comprises a nucleicacid for use in the invention for administration to a certain species ofanimal, the nucleic acid for use in the invention is preferably derivedfrom that species. For example, when the pharmaceutical composition isto be administered to humans, the nucleic acid of the pharmaceuticalpreferably comprises the soluble form of the human CD83 protein or aderivative thereof.

The nucleic acids for use in the invention can be administered inconjunction with agents that increase cell membrane permeability and/orcellular uptake of the nucleic acids. Examples of these agents arepolyamines as described for example by Antony, T. et al. (1999)Biochemistry 38:10775-10784; branched polyamines as described forexample by Escriou, V. et al (1998) Biochem. Biophys. Acta1368(2):276-288; polyaminolipids as described for example by Guy-Caffey,J. K. et al. (1995) J. Biol. Chem. 270(52): 31391-31396; DOTMA asdescribed by Feigner, P. L. et al. (1987) PNAS USA 84(21): 7413-7417 andcationic porphyrins as described for example by Benimetskaya, L. et al.(1998) NAR 26(23):5310-5317.

According to embodiment (15) of the invention the above defined solubleCD83 is suitable for preparing antibodies (polyclonal or monoclonal)against CD83. The antibodies can be prepared according to standardmethods known in the art. These antibodies are specifically useful forthe assay method of embodiment (16) and the kit (17) of the invention.Said assay method is specifically suitable for determining diseasescorrelated with an enhanced presence of soluble CD83 protein in thepatient's serum, preferably the method for determining tumor, autoimmunediseases, viral infections, etc., including B-Cell leukemia in apatient.

Using an Elisa test soluble CD83 was detected at a concentration ofapprox. 0.25 ng/ml (+/−0.25 ng/ml) in healthy individuals. Surprisingly,in tumor patients concentrations of up to 15 ng/ml were detected. Thus,this test could be of diagnostic and prognostic value for tumorpatients. In addition also for patients suffering from autoimmunedisorders, allergy and viral-, bacterial- and/or parasitic infections.

In the following, various aspects of the invention are more closelydescribed via examples. However, the invention should not be construedas being limited to the examples.

EXAMPLES Example 1 Recombinant Expression of Extracellular Human CD83Domain in Escherichia coli

Using a full-length human cDNA clone as a template, the extracellulardomain of CD83 was PCR-amplified (PCR conditions: 1 cycle of 1 min at94° C.; 30 cycles, each consisting of 1 min at 94° C. “denaturation”, 1min at 64° C. “annealing”, 1 min at 72° C. “extension”) using thefollowing PCR primers:

(SEQ ID NO.: 5) 5′-TCCCCGGGAACGCCGGAGGTGAAGGTGGCT-3′ (SEQ ID NO: 6)5′-AATTAGAATTCTCAAATCTCCGCTCTGTATT-3′.

The amplified cDNA fragment was cloned into the Smal and EcoRI sites ofthe expression vector pGEX2T (Amersham Pharmacia Biotech, Freiburg,Germany) resulting in the plasmid pGEX2ThCD83ext and this plasmid wastransformed into the E. coli strain TOP10F′ [F{lacI^(q)Tn10 (Tet^(R)}mcrA Δ (mrr-hsd RMS-mcrBC) φ 80 lcZ, Δ M15 Δ lacX74 recA1 deoR araD139 Δ(ara-leu) 7697 galU galK rpsL (Str^(R)) endA1 nupG] (Invitrogen,Groningen, The Netherlands). The correct nucleotide sequence ofpGEX2ThCD83ext was verified by sequencing. The extracellular CD83 wasexpressed as a fusion protein containing glutathione S-transferase as afusion partner at the amino-terminus. A thrombin cleavage recognitionsite was inserted between GST and the extracellular CD83 domain (SeeFIG. 1).

Example 2 Purification of the Recombinant Human CD83ext Cultivation:

An overnight bacterial culture of the above-mentioned bacteria wasdiluted 1:10 in fresh LB medium (supplemented with 100 i-ig/mlampicillin) and grown to an optical density of 1.0. IPTG was added(final concentration 1 mM) and the culture proceeded for a further hour.The cells were pelleted, resuspended in 10 ml native buffer (140 mMNaCI, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂ PO₄, 2. 6 mM MnCl₂, 26 mMMgCl₂, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml DNAse I, pH 7.6)per 500 ml culture and 50 μg/ml lysozym were added. After 15 minincubation on ice the lysate was centrifuged at 20,000×g.

Capture Step:

40 ml of the supernatant were added to a GSTrap 5 ml column on a ÄKTAExplorer 10 system (Amersham Pharmacia Biotech, Uppsala, Sweden) thatwas previously equilibrated with 4 column volumes of binding buffer: PBS(phosphate buffered saline), pH 7.6. The column was then washed with 12column volumes of the same binding buffer and subsequently eluted with 5column volumes of elution buffer: 50 mM Tris-HCl, pH 8.0 with 5 mMreduced glutathione at a flow rate of 5 ml/min. The column was thentreated with 5 column volumes of 2 M NaCl/PBS, pH7.6 and 5 columnvolumes of binding buffer (FIG. 2A).

Intermediate Purification Steps:

The GST-CD83ext-containing fractions were dialyzed against 50 mM1-methyl-piperazine (Sigma), 50 mM Bis-Tris (Sigma), 25 mM Tris (Sigma)pH9.5 (buffer A) and loaded onto a 15Q PE 4.6/100 anion exchange column(Amersham Pharmacia Biotech) on a ÄKTA Explorer 10 system (AmershamPharmacia Biotech). Proteins were separated by 3 different linear saltgradients: 16 column volumes to a target concentration of 10% buffer B(buffer A/1 M NaCl); 20 column volumes to a target concentration of 50%buffer B and 10 column volumes to a target concentration of 100% bufferB. (See FIG. 2B).

The GST-CD83ext-containing fractions were dialyzed against PBS, pH 7.6.Then, the GST-hCD83ext fusion protein was incubated with thrombin (20U/ml) on a glutathione-Sepharose matrix at 22° C. for 16 h. To separatethe hCD83ext protein from GST, this solution was loaded onto pre-packedglutathione-Sepharose 4B columns using the same buffer conditions as inthe capture step. Under binding buffer conditions, the flow-throughfraction containing recombinant human CD83ext protein was collected. Theresults are shown in FIG. 2C.

Polishing Step:

Finally, a preparative gel filtration separation was performed loadingthis flow-through fraction onto a Superdex 200 (26/16) prep grade column(Amersham Pharmacia Biotech) on a ÄKTA Explorer 10 system (AmershamPharmacia Biotech) using a running buffer of PBS, pH7.6, at a flow rateof 3 ml/min.

The correct fractions were tested by silver staining, coomassie stainingand Western blot analysis with anti-CD83 (Coulter-Immunotech,Marseilles, France) (See FIG. 2D).

Lyphilization of Recombinant Soluble CD83:

The HPLC purified recombinant soluble CD83 domain was dialysed against a1:20 dilution of DPBS (BioWhittaker Europe). Then this protein solutionwas frozen in liquid nitrogen and lyophilized for 4 h using an alpha 1-2LD freeze drying device (Christ). The protein was redissolved with 0.22μm filtered ddH₂O to a final concentration volume of 1×DPBS.

SDS Page analysis revealed that showed that the lyophilized recombinantprotein was not degraded after this procedure, in fact it was comparableto non-lyophilized protein (FIG. 2E).

Example 3 Inhibition of Dendritic Cell Maturation, In Vitro Cell Clusterand MLR Experiments (Human) Cultivation:

Unless otherwise noted, all cells were cultured using a standard medium(1% human plasma medium), which consisted of RPMI 1640 (BioWhittaker,Verviers, Belgium) supplemented with glutamine (200 μg/ml)(BioWhittaker, Verviers, Belgium), penicillin/streptomycin (20 μg/ml),10 mM Hepes, pH7.5 (Sigma-Aldrich), and 1% heat-inactivated (56° C.; 30min) human plasma from a single donor obtained from the Department ofTransfusion Medicine, Eriangen, Germany.

Generation of Dendritic Cells (DCs):

PBMCs were isolated from buffy coats by sedimentation in Ficoll-hypaque(Amersham Pharmacia Biotech, Freiburg, Germany) and seeded ontoIgG-coated (10 μg/ml γ-globulin from Cohn fraction; Sigma-Aldrich) 100mm-culture dishes and incubated at 37° C. in 5% CO₂. After 1 and 7 hincubations, non-adherent cell fractions were harvested, and theadherent cells were further cultured in 1% human plasma mediumsupplemented with the cytokines GM-CSF (800 U/ml) and IL-4 (500 U/ml).Fresh medium with GM-CSF to a final concentration of 400 U/ml and IL-4(500 U/ml) was added on day 3 of the incubation period. On day 4 or 5,non-adherent cells were collected, counted, and transferred into newdishes at a density of 0.3−0.5×10⁵ cells/ml. For final DC maturation, 1%human plasma medium was supplemented with TNF-α, (1.25 ng/ml), GM-CSF(40 U/ml), IL-4 (200 U/ml), prostaglandin E₂ (0.5 μg/ml). (Lechmann, M.et al. (2001) J. Exp. Med. 194:1813-1821).

Soluble hCD83ext Inhibits Maturation of Immature Dendritic Cells

To analyze the influence of hCD83ext on the phenotype of DC, FACSanalysis were performed on day 8 (See FIG. 3). DC can be fully maturedwith the use of a specific maturation cocktail composed of IL-1β, TNF-αand PGE₂ (FIG. 3 a). Interestingly, when this maturation cocktail wasadministered to immature DC on day 5 together with hCD83ext (4 μg/ml)and left until the final FACS analysis on day 8, these cells revealed aclear reduction in CD80 (from 96% to 66%) and CD83 cell surfaceexpression (96% to 30%) (FIG. 3 c), when compared with normally maturedDC (FIG. 3 a). Thus, hCD83ext induces a reduction in DC maturation (seealso increase of CD14 positive cells). In contrast, mature DC whichwhere incubated with hCD83 for 24 hours on day 7 and analyzed on day 8,showed only a minimal influence on CD80 expression (96% to 92%), whileCD83 expression was also reduced (96% to 66%) (FIG. 3 b). Interestingly,CD86 expression was not influenced at any time point by theadministration of hCD83ext. Also MHC class I and II expression was notaffected, neither in immature nor in mature DC (Lechmann, M. et al.(2001) J. Exp. Med. 194:1813-1821) (see FIG. 3).

Allogenic MLR:

CD4+ and CD84′ T cells were isolated from buffy coats (harvestednon-adherent cell fractions were incubated with neuramidase treatedsheep erythrocytes, collected by ficoll gradient centrifugation andcultured in RPMI, supplemented with 5% human serum from a single ABdonor) and stimulated with different ratios of mature allogenic DCs. Thecells were left untreated or were incubated with differentconcentrations of hCD83ext or with BSA (Biorad) as a control. T-cells(2×10⁵/well) and DCs were co-cultivated for 4 days in 200 [DJ RPMI,supplemented with 5% human serum from a single AB donor in 96-well cellculture dishes. Cells were pulsed with (³H]-thymidine (1 μCi/well;Amersham Pharmacia Biotech) for 16 h. The culture supernatants wereharvested onto glass fiber filtermates using an 1H-110 harvester(Inotech. Dottikon, Switzerland), and filters were counted in a 1450microplate counter (Wallac, Turku, Finnland) (Lechmann, M. et al. (2001)J. Exp. Med. 194:1813-1821).

A typical feature of these MLR-assays is the formation of large DC-Tcell-clusters. Addition of hCD83ext at day 1 strongly inhibited thetypical cell cluster formation of DC and proliferating T cells(Lechmann, M. et al. (2001) J. Exp. Med. 194:1813-1821).

Furthermore, mature dendritic cells treated with soluble hCD83ext areinhibited in a concentration dependent manner in their ability tostimulate T cell. Thus T cells do not proliferate anymore (See FIG. 4).

Example 4 In Vitro Cell Cluster and MLR Experiments (Mouse)

Male or female C57/BL6 mice and BALB/C mice (Charles River, Wiga,Sulzfeld, Germany) were used at the ages of between 1 and 4 months.

Generation of Bone Marrow (BM)-DCs:

The generation of BM-DCs from C67/BL6 mice was performed exactly asdescribed (J. Immunol. Methods 223:77, 1999). RPMI 1640 (LifeTechnologies, Karlsruhe, Germany) was supplemented with 100 U/mlpenicillin (Sigma), 100 ug/ml streptomycin (Sigma), 2 mM L-glutamine(Sigma), 50 μg/ml ME (Sigma), 10% heat-inactivated filtered FCS (PAA,Cölbe, Germany). GM-CSF was used at 200 U/ml (PrepoTech/Tebu, RockyHill, N.J.) on days 0, 3, 6 and 8 of incubation period.

Allogenic MLR:

CD4⁺ and CD8⁺ T cells were isolated from inguinal and mesentchymal lymphnodes of BALB/C mice and used for the allogenic MLR. These T-cells(2×20⁵ cells/well) and day 9 BM-DCs (at different ratios) wereco-cultured for 3 days in 200 μl RPMI 1640 supplemented with 100 U/mlpenicillin, 100 (μg/ml streptomycin, 2 mM L-glutamine, 50 μg/ml ME, 10%heat-inactivated filtered FCS in 96-well cell culture dishes. Cells werepulsed with [³H]-thymidine (1p Ci/well; Amersham Pharmacia Biotech) for16 h. The culture supernatants were harvested onto glass fiberfiltermates using an IH-110 harvester (Inotech, Dottikon, Switzerland),and filters were counted in a 1450 microplate counter (Wallac, Turku,Finnland).

Cluster formation between mouse dendritic cells and mouse T cells wasinhibited by soluble human hCD83ext. In addition, murine dendritic cellstreated with soluble human hCD83ext are inhibited in a concentrationdependent manner in their ability to stimulate T cell. Thus T cells donot proliferate anymore (See FIG. 5A).

Biological Activity of Lyophilized Recombinant CD83:

The biological activity of the lyophilized protein was determined by itsinhibitory activity in mixed lymphocyte reaction analysis as describedabove. The protein inhibits dendritic cell mediated T-cell stimulationin a dose dependent manner just like non-lyophilized protein (see FIG.5B). Thus, recombinant soluble CD83 is stable after freeze drying andkeeps its biological activity. A similar effect was observed in a humansystem (data not shown).

Example 5 Inhibition of Experimental Autoimmune Enzephalomyelitis (EAE)

EAE is the standard model for multiple sclerosis. EAE was induced inmice by injecting subcutaneosly into both tights 50 μl of a suspensioncontaining complete Freundsch'es adjuvans (CFA) and myelinoligodendrocyte glycoprotein (MOG₃₅₋₅₅) on day 0. On the same day 100 μlof pertussis toxin (2 μg/ml) were injected intraperitoneally. On day 2,a second dose of pertussis toxin was administered. Clinical signs ofparalysis appeared between days 10 and 14.

Inhibition of EAE in an In Vivo Model:

To test the ability of hCD83ext to prevent and suppress paralysisassociated with EAE, 100 μl hCD83 (1 μg/1 μl) were administered byinjection on days −1, 1 and 3 (See FIG. 6A). As control, one group ofmice was injected with 100 μl BSA (1 μg/1 μl). A third group of mice wasleft untreated. In all three groups of mice EAE was induced on day O,Surprisingly, hCD83ext almost completely inhibited the paralysisassociated with EAE.

Long Lasting Effect of EAE Inhibition:

It was shown that even when EAE is induced a second time, CD83 treatedmice are still protected (three doses of soluble CD83 protect mice fromEAE).

EAE was induced as described above: 100 μg of hCD83 (or BSA as control)were injected (i.p.) on day −1, 1 and 3. EAE was induced by subcutaneous(s.c.) injection of MOG peptide emulsified in CFA enriched with M.tuberculosis at day 0. In addition, 200 ng Pertussis toxin (Pt) wereadministered (i.p.) on day 0 and 2. hCD83 almost completely inhibitedthe paralysis, while BSA treated and untreated mice developed strongdisease symptoms (see FIG. 6B; 1.EAE, left panel). On day 28 EAE wasinduced a second time by immunizing the mice with MOG peptide asdescribed above. Strikingly, mice which were treated only three timeswith soluble CD83 were completely protected, while untreated andBSA-treated mice were paralyzed (see FIG. 6B; 2.EAE right panel).

Inhibition of EAE in a Therapeutic Application:

EAE was induced as described above by subcutaneous (s.c.) injection ofMOG peptide emulsified in CFA enriched with M. tuberculosis at day 0. Inaddition, 200 ng Pertussis toxin (Pt) were administered (i.p.) on day 0and 2. hCD83ext (100 μg/dose) was given 14 times, every second day, fromday 3 onwards. Even in this therapeutic setting soluble CD83 was able tostrongly influence the EAE symptoms. BSA (100 μg/dose) was used anegative control (see FIG. 6C).

Example 6 Production of Monoclonal Antibodies Against Human CD83

Approximately 50 μg of the GST-hCD83ext fusion protein was injectedintraperitoneally (ip) and subcutaneously (sc) into LOU/C rats. After a2 months interval, a final boost with the antigen was given ip and sc 3days before fusion. Fusion of the myeloma cell line P3X63-Ag8.653 withrat immune spleen cells was performed according to standard procedure.Hybridoma supernatants were tested in a solid-phase immunoassay usingthe GST-hCD83ext protein adsorbed to polystyrene microtiter plates.Following incubation with culture supernatants for 1 h, bound monoclonalantibodies were detected with peroxidase-labeled goat antirat IgG+IgMantibodies (Dianova, Hamburg, Germany) and o-phenylenediamine aschromogen in the peroxidase reaction. An irrelevant GST fusion proteinserved as a negative control. The immunoglobulin isotype of themonoclonal antibodies was determined using biotinylated antiratimmunoglobulin (IgG) subclass-specific monoclonal antibodies (ATCC,Rockville, Md.). CD83-1G11 (rat IgG1) and CD83-4B5 (rat IgG2a) were usedfor Western blot and FACS analysis.

Example 7 Determination of Soluble CD83 in Patients

Using an Elisa test soluble CD83 was detected at a concentration ofapprox. 0.25 ng/ml (+/−0.25 ng/ml) in healthy individuals. Surprisingly,in tumor patients concentrations of up to 15 ng/ml were detected. Thus,this test could be of diagnostic and prognostic value for tumorpatients.

Example 8 hCD83ext is a Disulfide-Linked Homodimeric Protein

The HPLC-purified recombinant human CD83ext protein (cloning andexpression as described in example 1, purification as described inexample 2 was analysed with the Laemmli SDS-PAGE system. To identifypossible oligomeric forms of CD83 2-mercaptoethanol (ME) has beenomitted from the sample buffer (2% SDS, 5% 2-Mercaptoethanol (ME), 10%Glycerol, 0.2 mM EDTA, 0.005% bromphenolblue, 62.5 mM Tris pH6.8). Inthe absence of this reducing agent, the intra- and interchain disulfidebonds of CD83 remain intact. The reduced and non-reduced protein sampleswere both incubated for 5 min at 95° C. and compared with each other bySDS-PAGE (see FIG. 7). During electrophoresis, the mobilities ofoligomeric SDS-proteins is lower than those of their fully denaturedSDS-polypeptide components. Without ME an upper band appears at theestimated size of a CD83-dimer (about 25 kDa), while the monomeric CD83band (about 14 kDa) is fainting. Westernblot analysis using theanti-CD83 antibody CD83-1G11 (Lechmann et al., Protein Expression andPurification 24:445-452 (Mar. 2, 2002)) confirmed the specificity of theprotein bands. Thus, hCD83ext is a disulfide-linked homodimeric protein.

The inhibitory activity of the isolated disulfide linked homodimericprotein was determined in MLR experiments described in Examples 3 and 4.It was found that the inhibitory activity of the isolated homodimer wasidentical to that described in Examples 3 and 4.

Example 9 Generation of a Mutant Form of Soluble CD83

Cloning of hCD83ext mut129 Cys to Ser Mutant in Escherichia coli

The mutant extracellular domain of human CD83 (amino acids 20-145) wasPCR-amplified using the following primer set:

sense-pGEX2ThCD83: 5′-TCCCCCCGGG AACGCCGGAG GTGAAGGTGG CT-3′ andantisense-CD83extra_mutantCtoS: 5′-AATTAGAATT CTCAAATCTC CGCTCTGTATTTCTTAAAAG TCTCTTCTTT ACGCTGTGCAG GGGAT-3′ (MWG-Biotech AG; SEQ ID Nos:11 and 12, respectively). The antisense primer inserts a g to cnucleotide transversion which leads to an amino acid exchange ofCystidin to Serin at the amino acid position 129 (see FIG. 8). The PCRconditions were: 5 min initial denaturation step at 94° C., 31 cycles: 1min denaturation at 94° C., 1 min annealing at 61° C., 2 min elongationat 72° C.; and a final 10 min elongation step at 72° C. The amplifiedcDNA fragment was subcloned into the SmaI and EcoRI sites of theexpression vector pGEX2T (Amersham Pharmacia Biotech) resulting in theplasmid pGEX2ThCD83ext_mut129_C to S and was transformed into the E.coli strain TOPO10 (Invitrogen). The correct nucleotide sequence wasverified by sequencing.Recombinant Expression of the hCD83ext mut129 Cys to Ser mutant proteinin Escherichia coli

The expression and purification of the mutant hCD83ext was performed asdescribed above for the recombinant hCD83ext protein:

An overnight bacterial culture was diluted 1:10 in fresh LB medium(supplemented with 100 μg/ml ampicillin). At an optical density of 0.9,1 mM IPTG was added and the culture proceeded for a further 1 h. Thenthe cells were pelleted and resuspended in 10 ml native buffer (140 mMNaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂ PO₄, 2.6 mM MnCl, 26 mMMgCl₂, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml DNaseI, pH 7.6) per500 ml culture. 50 μg/ml lysozyme were also added. After 15 minincubation on ice the lysate was spun at 20000 g. Protein purification:capture step: 40 ml supernatant were added to a GSTrap 5 ml column on anÄKTA Explorer 10 system (Amersham Pharmacia Biotech). Binding buffer:PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂ PO₄, pH7.6).Elution buffer: 50 mM Tris-HCl, pH 8.0 with 5 mM reduced glutathione.Flow rate: 5 ml. Chromatographic procedure: 4CV (column volumes) bindingbuffer, 40 ml supernatant, 12CV binding buffer, 5CV elution buffer, 5CV2N NaCl/PBS, pH7.6, 5CV binding buffer. Then the GST-hCD83ext. fusionprotein was incubated with thrombin 20 U/ml at 22° C. for 16 h. Toseparate the hCD83ext protein from GST, the elution was loaded againonto a GSTrap 5 ml column using the capture step buffer conditions.Under binding buffer conditions the flow through containing recombinanthuman CD83ext protein was collected.

The purified hCD83ext_mut129_Cys to Ser was compared to purifiedhCD83ext by SDS-PAGE (see FIG. 9). Under reducing as well as undernon-reducing conditions the mutant form of CD83 showed a stablemonomeric band at 14 kDa. This band is comparable to the hCD83extwildtype protein analysed under reducing conditions. Under non-reducingconditions no CD83-dimer could be detected with the mutant CD83 protein.So the 5^(th) carboxyterminal cysteine of the extracellular CD83 domainis necessary for the creation of homodimers. Westernblot analysisconfirmed specificity of the bands (data not shown). The inhibitoryactivity of the hCD83ext_mut129_C to S as tested in MLR experimentsdescribed in Examples 3 and 4 was comparable with that of the compoundtested in Examples 3 and 4.

Example 10 Soluble CD83 Inhibits Proliferation of Spleen CellsInhibition of Spleen Cell Proliferation:

Thirty, or alternatively sixty days after immunization of mice with MOG,spleens were removed for restimulation assays. Cells were cultured inHL-1 serum free medium supplemented with penicillin (100 U/ml, Sigma),streptomycin (100 μg/ml, Sigma), L-glutamin (2 mM, Sigma) and2.mercaptoethanol (50 μM, Sigma). MOG-specific cells were analyzed byincubating 4×10⁵ spleen cells with different concentrations of MOGpeptide in 200 μl HL-1/well in a 96-well tissue culture plate.Additionally, as a control, 4×10⁵ spleen cells were stimulated with IL-2(500 U/ml, Proleukin). As a negative control unstimulated cultures wereused. After 72 hours cultures were pulsed with [³H] thymidine (0.4Ci/mmol, Amersham TRA-20). Twelve hours later thymidine incorporationwas measured using a microplate counter (Wallac).

Spleen cells derived form hCD83ext treated mice show a clearly reducedproliferation (see FIG. 10A). Additionally, as a control, 4×10⁵ spleencells were stimulated with IL-2 (500 U/ml). Also hCD83ext treated cellsare still able to proliferate in response to IL-2 (see FIG. 10A, inserton the right hand site). These data clearly show that proliferation ofspleen cells is reduced in CD83 treated mice, however they can berestimulated using IL-2. Thus, they are not dead.

Restimulation of spleen cells derived from hCD83ext-, BSA- or un-treatedmice, where EAE was induced twice (see FIG. 10B). hCD83ext treated miceshow a slightly reduced proliferation capacity. However, while BSAtreated and untreated mice still strongly proliferate in response toIL-2, hCD83ext treated cells proliferate less in response to IL-2 (seeFIG. 10B, insert on the right hand site).

These data clearly show that proliferation of spleen cells is reduced inCD83 treated mice.

Example 11 Soluble CD83 Inhibits Cytokine Production by Spleen Cells

Harvested splenocytes which were stimulated with differentconcentrations of the MOG peptide (as described in Example 10), whereexamined regarding their ex vivo cytokine production. Culturesupernatants were taken after 96 hours and tested using commerciallyavailable sandwich ELISA kits for INF-γ, IL-2, IL-4, IL-10 (BDBiosciences). hCD83ext treated cells (after the first EAE induction) arestrongly inhibited in their IFN-γ production (see FIG. 11A). Also theIL-10 production is clearly reduced. IL-2 and IL-4 production are notdramatically influenced. These data clearly show that soluble CD83 leadsto a reduced cytokine production in the treated animals.

The cytokine production of spleen cells was also determined in spleencells derived from animals where EAE was induced twice (see FIG. 11B).IFN-γ production is strongly inhibited. The same is true for the IL-10production. IL-2 production is not greatly influenced. There is someIL-4 production in BSA- and un-treated cells, however the values arevery low and close to the detection limit. Again, these data clearlyshow that soluble CD83 leads to a reduced cytokine production in theanimals where EAE has been induced a second time.

1. A nucleic acid encoding a monomeric soluble CD83 protein selected from the group consisting of: i) a soluble CD83 protein consisting of amino acid residues 20 to 144 of SEQ ID NO: 2; ii) a soluble CD83 protein consisting of amino acid residues 20 to 145 of SEQ ID NO:2, iii) a soluble CD83 protein consisting of amino acid residues 1 to 129 of SEQ ID NO: 8; and iv) a soluble CD83 protein consisting of amino acid residues 1 to 130 of SEQ ID NO:8; wherein in each of i) through iv) the third or fifth cysteine residue is substituted with an amino acid residue selected from the group consisting of serine, alanine, glycine, valine, threonine, aspartic acid, glutamic acid, arginine, lysine, histidine, asparagine, glutamine and tyrosine; wherein said third cysteine residue corresponds to residue 100 of SEQ ID NO: 2 or residue 85 of SEQ ID NO: 8, and wherein said fifth cysteine residue corresponds to residue 129 of SEQ ID NO: 2 or residue 114 of SEQ ID NO:
 8. 2. The nucleic acid encoding a monomeric soluble CD83 protein of claim 1, wherein said cysteine residue of said monomeric soluble CD83 protein is substituted with serine.
 3. The nucleic acid encoding a monomeric soluble CD83 protein of claim 1, wherein said monomeric soluble CD83 protein consists of amino acid residue 20 to 145 of SEQ ID NO:2 and wherein the third or fifth cysteine residue is substituted with an amino acid residue selected from the group consisting of serine, alanine, glycine, valine, threonine, aspartic acid, glutamic acid, arginine, lysine, histidine, asparagine, glutamine and tyrosine.
 4. The nucleic acid encoding a monomeric soluble CD83 protein of claim 1, wherein said monomeric soluble CD83 protein consists of amino acid residues 1 to 130 of SEQ ID NO:8, and wherein the third or fifth cysteine residue is substituted with an amino acid residue selected from the group consisting of serine, alanine, glycine, valine, threonine, aspartic acid, glutamic acid, arginine, lysine, histidine, asparagine, glutamine and tyrosine.
 5. The nucleic acid encoding a monomeric soluble CD83 protein of claim 3, wherein the third cysteine residue of said monomeric soluble CD83 protein has been substituted with a serine residue.
 6. The nucleic acid encoding a monomeric soluble CD83 protein of claim 1, wherein the third cysteine residue of said monomeric soluble CD83 protein, corresponding to residue 100 of SEQ ID NO:2 or residue 85 of SEQ ID NO:8, is substituted.
 7. The nucleic acid encoding a monomeric soluble CD83 protein of claim 1, wherein the fifth cysteine residue of said monomeric soluble CD83 protein, corresponding to residue 129 of SEQ ID NO:2 or residue 144 of SEQ ID NO:8, is substituted.
 8. The nucleic acid encoding a monomeric soluble CD83 protein of claim 1, which consists of amino acid residues 1 to 130 of SEQ ID NO:10.
 9. The nucleic acid encoding a monomeric soluble CD83 protein of claim 4, wherein the third cysteine residue, corresponding to amino acid residue 85, is substituted with an amino acid residue which is serine.
 10. The nucleic acid encoding a monomeric soluble CD83 protein of claim 6, wherein the third cysteine residue of said monomeric soluble CD83 protein is substituted with a serine residue.
 11. The nucleic acid encoding a monomeric soluble CD83 protein of claim 7, wherein the fifth cysteine residue of said monomeric soluble CD83 protein is substituted with a serine residue.
 12. A nucleic acid comprising a start codon operatively linked to a sequence encoding a monomeric soluble CD83 protein of the CD83 family of proteins selected from the group consisting of: i) a soluble CD83 protein consisting of amino acid residues 20 to 144 of SEQ ID NO: 2; and ii) a soluble CD83 protein consisting of amino acid residues 20 to 145 of SEQ ID NO: 2; wherein in each of i) and ii) the third or fifth cysteine residue is substituted with an amino acid residue selected from the group consisting of serine, alanine, glycine, valine, threonine, aspartic acid, glutamic acid, arginine, lysine, histidine, asparagine, glutamine and tyrosine; wherein said third cysteine residue corresponds to residue 100 of SEQ ID NO: 2, and wherein said fifth cysteine residue corresponds to residue 129 of SEQ ID NO:
 2. 13. The nucleic acid of claim 12, wherein said sequence consists of amino acid residues 20 to 144 of SEQ ID NO:2, and wherein the third cysteine residue of said encoded monomeric soluble CD83 protein, corresponding to residue 100 of SEQ ID NO:2, is substituted with a serine residue.
 14. The nucleic acid of claim 12, wherein said sequence consists of amino acid residues 20 to 145 of SEQ ID NO:2, and wherein the fifth cysteine residue of said encoded monomeric soluble CD83 protein, corresponding to residue 129 of SEQ ID NO:2, is substituted with a serine residue.
 15. A host cell comprising the nucleic acid of claim
 12. 16. The host cell of claim 15, wherein said cell is Escherichia coli.
 17. A protein encoded by the nucleic acid of claim
 12. 18. The protein of claim 17, wherein the protein has a native glycosylation pattern.
 19. A protein encoded by the nucleic acid of claim
 14. 20. A pharmaceutical composition comprising the monomeric soluble CD83 protein of claim
 17. 21. A method for producing a soluble CD83 protein comprising culturing the host cell of claim
 15. 22. The method of claim 21, wherein said host cell is a prokaryotic host cell which is Escherichia coli.
 23. The nucleic acid of claim 12, wherein said sequence consists of amino acid residues 20 to 145 of SEQ ID NO:2, and wherein the third cysteine residue of said encoded monomeric soluble CD83 protein, corresponding to residue 100 of SEQ ID NO:2, is substituted with a serine residue.
 24. A protein encoded by the nucleic acid of claim
 23. 